Proteus mirabilis is known to be highly resistant to the action of polymyxin B (PB). However, the mechanism underlying PB resistance is not clear. In this study, we used Tn5 transposon mutagenesis to identify genes that may affect PB resistance in P. mirabilis. Two genes, ugd and galU, which may encode UDP-glucose dehydrogenase (Ugd) and UDP-glucose pyrophosphorylase (GalU), respectively, were identified. Knockout mutants of ugd and galU were found to be extremely sensitive to PB, presumably because of alterations in lipopolysaccharide (LPS) structure and cell surface architecture in these mutants. These mutants were defective in swarming, expressed lower levels of virulence factor hemolysin, and had lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd or galU gene, respectively, led to the restoration of wild-type phenotypic traits. Interestingly, we found that the expression of Ugd and GalU was induced by PB through RppA, a putative response regulator of the bacterial two-component system that we identified previously. Mutation in either ugd or galU led to activation of RpoE, an extracytoplasmic function sigma factor that has been shown to be activated by protein misfolding and alterations in cell surface structure in other bacteria. Activation of RpoE or RpoE overexpression was found to cause inhibition of FlhDC and hemolysin expression. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.
Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (>160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg 2؉ . Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.
Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.
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